Identification and quantitation of residual host cell proteins in protein samples

Abstract

The present disclosure relates to highly sensitive methods for determining the identity and quantity of one or more proteins in a sample. For example, the present disclosure provides methods for the highly sensitive identification and quantitation of residual host cell proteins in protein samples and can be adapted to identify and quantify proteins in either a targeted or a target-agnostic manner and can be modified to achieve a range of sensitivities appropriate for distinct use cases.

1 . A method for identifying one or more proteins in a sample comprising a protein product, comprising:
 a) contacting a sample comprising one or more proteins with a protease under conditions sufficient to digest proteins present in the sample;   b) contacting the sample comprising the digested protein with sodium deoxycholate (SDC) under reducing and heated conditions;   c) contacting the sample comprising the digested protein to a chromatographic support to remove undigested protein;   d) contacting the chromatographic support with a mobile phase and collecting an eluent; and   e) analyzing a fraction of the eluent using liquid chromatography/mass spectrometry analysis (LC-MS/MS) to identify one or more proteins in the sample.   
     
     
         2 . The method of  claim 1  wherein the LC-MS/MS is performed in data dependent acquisition (DDA) mode. 
     
     
         3 . The method of  claim 1 , wherein the protein is a host cell protein. 
     
     
         4 .- 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the w/w ratio of protease-to-protein product in the sample is about 1:2000, about 1:800, about 1:400, or about 1:200. 
     
     
         8 . The method of  claim 1 , wherein the digested protein sample is contacted with SDC at about 0.9% w/v to about 1% w/v. 
     
     
         9 .- 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the method has a limit of detection (LOD) from about 0.1 ppm to about 5 ppm. 
     
     
         12 . The method of  claim 1 , wherein the protein product is an antibody. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the sample comprising the protein product is a partially or fully purified sample. 
     
     
         15 . The method of  claim 14 , wherein the partially or fully purified sample is:
 (i) an ultrafiltration/diafiltration pool sample,   (ii) a drug substance sample; or   (iii) a drug product sample.   
     
     
         16 .- 17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein a load of the protein product in the sample is from about 6 μg to about 300 μg. 
     
     
         19 . The method of  claim 1 , wherein:
 (i) the contacting step (a) is for a time from about 2 to about 4 hours and/or   (ii) the contacting step (a) is performed at about 37° C.; and/or   (iii) the contacting step (b) is for about 10 minutes; and/or   (iv) the contacting step (b) is performed at about 90° C.   
     
     
         20 .- 22 . (canceled) 
     
     
         23 . A method for determining the ppm level of one or more target proteins in a sample comprising a protein product, comprising:
 a) contacting the sample with a protease under conditions sufficient to digest the one or more target proteins;   b) contacting the sample comprising the one or more digested target proteins with SDC under reducing and heated conditions;   c) contacting the sample comprising the one or more digested target proteins to a chromatographic support to remove undigested protein;   d) contacting the chromatographic support with a mobile phase and collecting an eluent; and   e) analyzing the eluent using LC-MS/MS to identify and quantitate the one or more target protein in the sample, wherein said analysis comprises determining the signals associated with a plurality of standard ppm levels of the one or more target protein and comparing those signals to the signal detected for the one or more target protein in the sample.   
     
     
         24 . The method of  claim 23 , wherein the LC-MS/MS is performed in parallel reaction monitoring (PRM) mode. 
     
     
         25 .- 28 . (canceled) 
     
     
         29 . The method of  claim 23 , wherein the w/w ratio of protease-to-protein product in the sample is about 1:2000, about 1:800, about 1:400, or about 1:200. 
     
     
         30 . The method of  claim 23 , wherein the one or more digested target protein sample is contacted with SDC at about 0.9% w/v. 
     
     
         31 .- 32 . (canceled) 
     
     
         33 . The method of  claim 23 , wherein the method has a limit of quantitation (LOQ) of about 0.01 ppm. 
     
     
         34 . The method of  claim 23 , further comprising normalization of data from the LC-MS/MS analysis. 
     
     
         35 . The method of  claim 23 , wherein the protein product is an antibody. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 23 , wherein the sample comprising the protein product is a partially or fully purified sample. 
     
     
         38 . The method of  claim 37 , wherein the partially or fully purified sample is:
 (i) a ultrafiltration/diafiltration pool sample;   (ii) a drug substance sample; or   (iii) a drug product sample.   
     
     
         39 . (canceled) 
     
     
         40 . The method of  claim 23 , wherein a load of the protein product in the sample is from about 6 μg to about 300 μg. 
     
     
         41 . The method of  claim 23 , wherein:
 (i) the contacting step (a) is for a time from about 2 to about 4 hours;   (ii) the contacting step (a) is performed at about 37° C.; and/or   (iii) the contacting step (b) is for about 10 minutes; and/or.   (iv) the contacting step (b) is performed at about 90° C.   
     
     
         42 .- 66 . (canceled) 
     
     
         67 . The method of  claim 11 , further comprising adjusting the sample load of the protein product to achieve the LOD from about 0.1 ppm to about 5 ppm. 
     
     
         68 . The method of  claim 1 , wherein the fraction of the eluent contains about 6 μg, about 30 μg, about 60 μg, about 150 μg, or about 300 μg of protein product. 
     
     
         69 . The method of  claim 1 , wherein analyzing step (e) further comprises normalization of data from the LC/MS analysis.